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. 2012 May 23;3(5):518–524. doi: 10.18632/oncotarget.500

Figure 2. TGFβ stimulates c-cbl-dependent Lyn ubiquitination.

Figure 2

(A) TGFβ induces Lyn ubiquitination. HEK293T cells were co-transfected with expression plasmids for Lyn and His-tagged Ubiquitin. Cells were put into 2% FCS-containing media overnight before TGFβ treatment (1ng/ml) for a range of time between 0-360 minutes, or 10μM SB431532. Cell lysates were incubated overnight with nickel-NTA beads, and bound proteins eluted with Laemmli buffer. Samples were separated by SDS-PAGE and Western blots probed with anti-His, anti-Lyn or anti-actin antibodies as indicated. (B) HEK293T cells were transfected with Lyn, c-Cbl and Ub plasmids as indicated. Cells were maintained in 2% FCS media post-transfection before TGFβ (1ng/ml) or SB431542 (10μM) stimulation for 60 minutes. Lysates were incubated with Nickel-NTA beads, and bound proteins eluted with Laemmli buffer. Samples were separated by SDS-PAGE and Western blots probed with anti-His, anti-Lyn or anti-actin antibodies as indicated. (C) MYL cells were stimulated with TGFβ (1ng/ml) for 60minutes. RNA was isolated, reverse transcribed, and and 5ng cDNA utilised as template DNA in qRT-PCR using primers specific for c-cbl. Data represent triplicate samples, and are normalised against 18S RNA as an internal control.